The ability to avoid replicative senescence is a hallmark of malignant transformation. At least 85% of all cancers and 90% of breast cancers acquire this capability by activating telomerase, a reverse transcriptase that uses an internal telomerase RNA template to extend chromosome ends with telomeric DNA repeats. For this reason, telomere destabilizers and telomerase inhibitors have been a central focus of anti-tumor chemotherapy. Yet, due to a limited understanding of the basic cellular processes that cause telomere fragility and due to high background noise in current cell-based assays for telomerase inhibitor screens, there has been minimal discovery of clinically relevant telomere and telomerase targeting agents. This is a study of how variant telomerase RNA templates can be used to reveal non-WT telomere sequences that are either compatible or incompatible with cellular survival. The overall hypothesis is that randomization of the template will result in the synthesis of one class of telomeric sequences that maintain core telomere functions and thereby support cell viability and a separate class that rapidly becomes uncapped by protective proteins and elicits a potent DNA Damage Response (DDR) that confers cell death. The goal of Aim 1 is to define the mechanism by which features of the Schizosaccharomyces pombe telomerase RNA (TER1) template and complementary telomere sequence can cause cellular senescence. Serial restreaks and fluorescence microscopy of DDR and repair factors will be used to achieve this aim. The resultant information will guide therapeutic strategies to uncap telomeres in cancer cells. The purpose of Aim 2 is to identify alternative TER1 sequences that can serve as a template for the synthesis of variant telomeric repeats while retaining physiologic telomere functions. The direct primer extension assay and chromatin immunoprecipitation (ChIP) will be employed to evaluate the recruitment and overall activity of variant telomerases. The ability of the telomeric proteins, Taz1 and Pot1, to bind alternative repeats will be assessed using in vitro biochemical assays coupled with ChIP. Physiologic telomere functions including protection against 3' end resection, chromosome fusions, and DDR signaling as well as gene silencing by the telomere position effect will be characterized. These experiments provide proof-of-principle that a competitive growth screen of randomized templates can reveal functional templates other than WT. Aim 3 applies this concept to human breast cancer cell lines and uses flow cytometry and Illumina sequencing to address which variant human telomerase RNA (hTR) templates confer a growth advantage versus apoptosis. The identification of alternative templates that correspond to telomere sequences distinct from WT will facilitate the development of cell-based assays that can use the synthesis of non-WT telomeres as a clear signal for telomerase activity in a background of WT telomeres.